The overall objective of this K01 proposal is to determine the significance of visceral adipose tissue (VAT) derived microRNAs (miR) in the genesis of systemic insulin resistance (IR) associated with obesity and type 2 diabetes mellitus (DM2). In this proposal, we posit a key role for miR-223 in the pathogenesis of obesity/DM2. Our overarching hypothesis is driven by three key sets of preliminary data: a) miR-223 is the most upregulated miR in VAT of humans, correlating strongly with markers of IR and inflammation, b) miR-223 regulates multiple targets involved in insulin signaling, and c) plasma exosomes derived from obese/IR patients are enriched in miR-223 compared to controls. We propose to test the significance of visceral adipose macrophages (VAT-) miR-223 as part of an ongoing inter-disciplinary investigation that will significantly enhance my training and propel my career towards independence. In Aim 1, we will compare miR-223 expression in VAT- with plasma exosome-miR-223 levels in obese patients undergoing bariatric surgery vs. non-obese controls, following post- surgery/post-weight loss exosome miR-223 levels compared to markers of BMI, IR, etc. We hypothesize that VAT- and exosome miR-223 strongly correlate with markers of glycemic control, IR, and inflammation. Next, we will test the in vitro effects of miR-223 overexpression on target genes relevant to IR as predicted by computational bioinformatics. In Aim 2, we hypothesize that VAT- are an important source of plasma exosome miR-223. We will assess the temporal course of miR-223 expression in various compartments [VAT- , liver (Kupffer cells), and plasma exosomes] and further hypothesize that exosome-miR derived from VAT- may contribute to regional (paracrine) and endocrine (liver) disruption of insulin signaling. To investigate this, we will assess in vitro transport of fluorescently labeled macrophage-derived exosomes containing miR-223 to human hepatocytes and adipocytes in co-culture assays. The effect of exosome-miR-223 on validated targets in human hepatocytes and adipocytes will be assayed. Finally in Aim 3, we hypothesize that miR-223 antagonism in VAT- accomplished via regional delivery of targeted antagomiR-223 nanoparticles will decrease functional plasma exosome miR-223, beneficially impacting whole body insulin sensitivity. Collectively, the experimental findings from this proposal should help drive new understanding of VAT-derived miRs as pleiotropic modulators of processes in IR/DM2.